By studying nerves in "pre-tadpole" frogs, researchers at the Johns Hopkins Institute for Cell Engineering have uncovered the first link between two key biological factors that guide growing nerves.

The finding sheds light on how nerves grow in the right direction so they can connect to the right places -- critical information to have if damaged nerves are ever to be repaired in people. In particular, the discovery reveals for the first time how the guidance cues that attract or repel the tip of a growing nerve influence the flow of calcium ions into the nerve cell, solving a decades-old mystery.

"For 20 years researchers have known that calcium flow is critical for proper nerve growth, but no one has known how it gets into the nerve in response to a guidance cue," says Guo-Li Ming, M.D., Ph.D., assistant professor of neurology in the Institute for Cell Engineering's Neuroregeneration and Repair Program. "Now we have some details about how that happens in frogs. The findings are likely to hold for other animals and people, too, because we have similar versions of these proteins."

In the June issue of Nature Neuroscience, the Hopkins team shows that a "dual-action" protein connects guidance cues and the calcium flows that allow the nerve to respond to those cues. This protein, called TRPC1 or transient receptor potential channel 1, lets calcium into the nerve's growing tip once it is "turned on" by certain guidance cues. Furthermore, in pre-tadpole-stage frogs, TRPC1 is required for a specific set of nerves in the spinal cord to grow properly.

The Johns Hopkins scientists' research confirms a recent report that laboratory-grown frog nerves rely on TRPC1 activity to help direct the nerves' axons, and it shows for the first time that the protein is required for normal nerve growth in the actual frog.

"Frogs have only TRPC1, but humans have a whole family of TRPC proteins, including several analogous to the frog's TRPC1," says Ming. "It's likely that the same role is being played by these proteins during human development."

But TRPC1 also may play a role later in life, when guidance cues in the brain and spinal cord tell nerves not to regrow if they are damaged. Understanding exactly how these cues influence nerve cells could help efforts to overcome their signals and get damaged nerves to regrow their axons and reform lost connections.

"We also found that TRPC1 triggers calcium influx in response to myelin-associate glycoprotein, a molecule that helps prevent the regrowth of damaged nerves in adults," says Ming. "If this is also true in people, it could be critical to trying to get around myelin's growth-inhibiting effects."

The researchers' experiments also show that TRPC1 is used for detecting and reacting to attractive guidance cues netrin-1 and brain-derived neurotrophic factor during development. There are likely to be many others, the scientists say.

TRP channels were first identified by Craig Montell, professor of biological chemistry and neuroscience at Johns Hopkins, and were already known to help cells sense heat and pain.

"Now we've shown that they also are likely to be critical for proper nerve growth during embryonic development and to be part of the machinery that prevents nerve regrowth later in adulthood," says study co-author Hongjun Song, Ph.D., also an assistant professor of neurology in ICE's Neural program.

Situated in the spinal cord of the frog, the nerves affected by TRPC1 use it at least in part to extend their tentacle-like axons along the circumference of the spinal cord toward and then across the cord's midline. Each axon then branches and shoots one branch along the length of the spinal cord toward the brain and the other "down" toward the animal's legs.

To study TRPC1's effects in frogs, the researchers used a number of different methods to interfere with the ability of one side of the animal's spinal cord to react to the protein. In one set of experiments, the researchers injected the equivalent of interfering RNA (DNA's cousin) into one cell when the developing frog was at just a two-cell stage. Because each of the cells develops into half the frog, one side is normal, and one side can't make sufficient amounts of TRPC1.

The researchers discovered that interfering with TRPC1 prevented many of the growing nerves from reaching or crossing the midline of the spinal cord in the developing frogs.

"This is a major breakthrough in understanding how calcium gets into these cells in response to guidance cues during development," says Song.

Authors on the paper are Sangwoo Shim, Eyleen Goh, Shaoyu Ge, Kurt Sailor, Joseph Yuan, Paul Worley, Song and Ming, all of Johns Hopkins; and Llewelyn Roderick and Martin Bootman of the Babraham Institute, Cambridge. The Hopkins researchers were funded by the National Institute of Neurological Disorders and Stroke, Charles E. Culpeper Scholarships in Medical Science, the Whitehall Foundation, and the Basil O'Connor Starter Scholar Research Award Program to Guo-li Ming. Sangwoo Shim is partially supported by a postdoctoral fellowship from the Korea Science and Engineering Foundation.